1 Volume 21 2022 e226427 Original Research Braz J Oral Sci. 2022;21:e226427http://dx.doi.org/10.20396/bjos.v21i00.8666427 1 Department of Odontology, Federal University of Bahia, Salvador, Bahia, Brazil. 2 Department of Odontology, Federal University of Sergipe, Aracaju, Sergipe, Brazil. 3 Department of Morphology, Federal University of Sergipe, Sergipe, Brazil. 4 Department of Oral Surgery and Pathology, Dentistry Faculty of Federal University of Minas Gerais, Minas Gerais, Brazil. Corresponding author: Liciane dos Santos Menezes Address: Department of Odontology, Federal University of Bahia, Avenida Reitor Miguel Calmon s/n - Vale do Canela, 4º andar (Sala 404), Salvador, Bahia, Brazil, 40110-902 E-mail: licianesmenezes@gmail.com Phone number: (+55 79) 998985676 Editor: Altair A. Del Bel Cury Received: July 21, 2021 Accepted: February 25, 2022 Mutagenicity in oral cells of individuals exposed to radiofrequency generated by different smartphones Liciane dos Santos Menezes1,* , Itana Silva Santos2 , Marcos Antônio Lima dos Santos2 , Andrea Ferreira Soares3 , Sílvia Ferreira de Sousa4 , Wilton Mitsunari Takeshita2 Aim: This study aimed to investigate whether non-ionizing radiation emitted by smartphones is likely to cause genotoxic effects on oral epithelial cells. Methods: Thirty adults were distributed into two groups according to the mobile phone brand used, namely Samsung (Samsung, Seoul, South Korea) and Apple (Apple, California, USA). The material was collected with gentle swabbing of the right and left buccal mucosa using a cervical brush, then the micronucleus test was performed. Results: The Mann-Whitney test with a 5% significance level did not reveal statistically significant differences in micronuclei frequency between the exposed and non-exposed sides (p=0.251). The different brands do not seem to cause risks of inducing genetic damage because there were no statistically significant differences between them (p=0.47). Conclusion: Therefore, our results suggest no correlations of micronuclei frequency in the exposed buccal cells of mobile phone users at the exposure standard levels observed. Keywords: Radio waves. Micronucleus tests. Mutagenicity tests. https://orcid.org/0000-0002-4971-2354 https://orcid.org/0000-0003-3194-5894 https://orcid.org/0000-0002-7589-4809 https://orcid.org/0000-0002-1442-6462 https://orcid.org/0000-0001-7820-4749 https://orcid.org/0000-0001-5682-1498 2 Menezes et al. Braz J Oral Sci. 2022;21:e226427 Introduction The advent of globalization and the importance of communication networks in this context has increased the use of technologies such as smartphones exponentially all over the world. More than two-thirds of the world population, meaning over five billion people, are mobile telephony subscribers. This technology is based on the exchange of signals between smartphones and base stations through electromag- netic waves known as radiofrequency electromagnetic fields (RF-EMF)1,2. Interna- tional organizations are responsible for establishing guidelines on limits of expo- sure to radiofrequency. According to the International Commission on Non-Ionizing Radiation Protection3 (2009), the acceptable limit, which is used in several coun- tries, is a maximum of 300 GHz. The rate at which RF-EMF is absorbed by the human body is called specific absorption rate (SAR), which is a standardized unit that measures the impact of radiofrequency electromagnetic waves on the human body and is expressed as Watt/kg. The maximum legal SAR level limited to any mobile phone is 1.6 Watt/kg2. Therefore, it is important to investigate the effects of exposure to these radiofrequency values. Radiofrequency exposure limits are necessary because the human body absorbs a part of electromagnetic waves, implying serious biological risks. Biological con- sequences such as a higher risk of neurological and auditory diseases have been reported in the literature but the results are contradictory4,5. The effects of these radiations are classified into thermal and non-thermal6. The first case happens because non-ionizing radiation can release a sufficient amount of energy to warm up the biological tissue, and serious damage may occur when exceeding the limit levels6,7. According to Christ and Kuster8 (2005), several factors influence the amount of radiation absorbed by the head of users, namely the power required to transmit and receive the signal from the radio base station (tower), the model of the antenna, phone design, and the positioning relative to the head. The energy emitted by RF-EMF is not sufficiently capable of causing direct DNA dam- age but may interfere with the genome through indirect mechanisms such as the production of reactive oxygen species, chromatin disorganization, and impairment of DNA repair9. Given the carcinogenic potential of RF-EMF in human cells, classified as a Group 2B agent10, the study of potential mutagenic alterations in the oral epithelium of individuals exposed to this radiation is relevant. De Oliveira et al.4 (2017) reported that DNA damage can trigger important cellular changes such as senescence, death, or malfunction. These genotoxic changes can be diagnosed with methods such as the micronucleus (MN) test, which allows observing chromatin fragments from chromosomal breakage due to clastogenic or aneugenic events also classified as genotoxicity biomarkers11. The MN test, performed through exfoliative cytology of the oral epithelium, is a useful and minimally invasive diagnostic tool for assessing genetic damage in humans12. According to Ros-Llor et al.13 (2012), the test is fast and practical, considering that oral mucosa cells are easier to collect than others, such as blood cells. 3 Menezes et al. Braz J Oral Sci. 2022;21:e226427 Considering the growing number of smartphone users, the risk of genetic damages may contribute to implementing measures to help eradicate long-standing miscon- ceptions about the radiation emitted by these devices. Thus, the present study aimed to evaluate, by microscopic observation using the MN test, whether non-ionizing radiation emitted by mobile phones of different brands caused mutagenic effects on oral mucosa cells. Materials and Methods Study population Thirty adults (12 men and 18 women) were selected for the study. The sample size calculation was based on the studies by Daroit et al.5 (2015), Souza et al.9 (2014), and Yadav and Sharma14 (2008), in which the mean and standard deviation values of 3.75 and 3.791, respectively, were used for the variable of “total micronucleus”. Consider- ing a study with 90% power and α = 0.05, the acceptable sample size was at least 13 participants in each group. Volunteers who reported not using a mobile phone or having no preference of side (right and left) when using the device were excluded. Individuals who reported using non-traditional mechanisms to answer calls such as headsets, hands-free devices, and Bluetooth were not included in the survey. Other exclusion criteria were the presence of diseases such as diabetes and anemia; use of medications; reports of facial trauma; pregnancy; chronic use of alcohol or drugs; smoking; use of ortho- dontic appliances; exposure to oral X-rays one month before the study; use of mouthwashes, and use of tooth-desensitizing or bleaching agents 21 days before the study. Volunteers with the following characteristics were included in the study: male or female individuals aged between 20 and 30 years and individuals with good general and oral health without changes in the oral mucosa. All participants signed the informed consent, and the study was approved by the Human Research Ethics Committee (CAAE: 53233716.5.0000.5546), following the Declaration of Helsinki. The participants included responded to a questionnaire on sociodemographic data, past medical history, family history, habits (e.g., alcohol and tobacco consumption), diet, and history of exposure to RF-EMF (time using smartphones, the number of minutes a day using smartphones, and side of the face preferred when using the device). The groups were divided according to the questionnaire answers and two mobile phone brands were compared: Samsung (head SAR value = 0.52 W/kg; body SAR value= 0.99W/kg) (Samsung, Seoul, South Korea) and Apple (head SAR value = 1.2W/kg; body SAR value= 1.13W/kg) (Apple, California, USA). Collection of material After a mouth rinse with water, cells were collected by gentle swabbing of the right and left buccal mucosa with a Cytobrush cervical brush (Adlin, Jaraguá do Sul/SC-Brazil). The cells were transferred to a vial containing a fixative solution 4 Menezes et al. Braz J Oral Sci. 2022;21:e226427 (Sra Medical, Balneário Camboriú/SC-Brazil). Then, they were homogenized in a vortex shaker at speed four for 30 seconds (NI 1059 - Novainstruments Equipa- mentos para Laboratórios Ltda., Piracicaba/SP-Brazil), centrifuged for 10 minutes at 1000 rpm, 130 × g (Baby I 206 – FANEM, Guarulhos/SP-Brazil), and finally placed on glass slides and allowed to dry at room temperature for about one hour. The cells were fixed on the glass slides with 80% ethanol for 48 hours before staining. After drying, the slides were stained with hematoxylin-eosin (HE), an acid-basic stain that produces a contrast between the cytoplasm and the nucleus. First, the samples were exposed to hematoxylin, a basic dye that binds to substances containing acid groups. Then, the samples were exposed to eosin, a weak acid colorant that stains basic structures. Considering this characteristic, HE has a high affinity with nuclear cells presenting great blue and pink colorations15. Analysis of slides An oral pathologist with over 10 years of experience performed a blind evaluation. Calibration was performed with the joint analysis of five slides, totaling approximately 6,000 cells. The intraclass correlation coefficient (ICC) value was 0.79, indicating excellent agreement. An Olympus CX31 transmitted light microscope model (São Paulo/SP-Brazil) was used for slide analyses. The slides were analyzed from left to right and top to bottom with a 40× objective. Then, an immersion objective was used for micronucleus anal- ysis. Micronuclei were searched in 2,000-cell nuclei per cytological smear15,16, and an additional 2,000 cells were analyzed when the frequency of micronuclei was higher than 2%. The micronuclei were identified according to the criteria by Sarto et al.17 (1987) for measuring DNA damage/genotoxicity. Data analysis The results of the microscopic analysis of the cell counts of the oral mucosa exposed to radiofrequency radiation were tabulated in Microsoft Excel, version 2010 for Win- dows 64-bit (Microsoft Corporation, Redmond, WA, USA). The Shapiro-Wilk test was used to verify the normality of distribution. As a non-Gaussian distribution was found, the Mann-Whitney test was used. A t-test was used to compare the mobile phone brands, as Gaussian distribution was observed in this case. The statistical tests were performed using the R software with the Rcmdr package, version 3.2.1 for Windows 64-bit (The R Foundation, Vienna, Austria). A 5% significance level was set for all statistical analyses. Results Cells of the right and left buccal mucosa of 30 individuals (15 users of Apple and 15 users of Samsung mobile phones) were evaluated, resulting in 60 samples. Table 1 shows the most important characteristics of the study population. There were male (40%) and female (60%) participants aged between 20 and 30 years. The total period of exposure in this study was predominantly in the range of over 10 years (73%). 5 Menezes et al. Braz J Oral Sci. 2022;21:e226427 Table 1. Characteristics of the study population. Patients: n 30 Age: mean 23.93 Sex: n (%) Male 12 (40) Female 18 (60) Time of exposure to mobile phones: n (%) < 5 years 0 (0) 5 – 10 years 8 (27) > 10 years 22 (73) Mobile phone use (h/week): n (%) 0 0 0 - 2 26 (87) 2 - 4 4 (13) Hand used to answer calls: n (%) Right 28 (93) Left 2 (7) According to the Mann-Whitney test, the micronuclei count was not statistically dif- ferent between exposed and non-exposed sides (p = 0.251) (Figure 1). Differences between brands were not statistically significant (Table 2). 20 18 16 14 12 10 8 6 4 2 0 p = 0.251 Exposed buccal mucosa Non-exposed buccal mucosa M ic ro nu cl ea te d ce lls /1 00 0 ce lls Figure 1. Median micronuclei count (maximum and minimum) regardless of brand (Mann-Whitney test). 6 Menezes et al. Braz J Oral Sci. 2022;21:e226427 Table 2. Micronuclei count according to mobile phone brand (t-test). Micronuclei Mean SD p-value Apple 2.70 ±1.45 0.47 Samsung 3.23 ±2.46 SD: standard deviation. Discussion In the global communication era, mobile phones are often used and some assump- tions regarding their side effects are questioned. The present study aimed to evaluate, with the MN assay, whether the radiation emitted by mobile phones can cause muta- genic effects on oral epithelium cells. The results presented in this study suggest that ionizing radiation associated with mobile phones does not induce the formation of micronuclei in buccal cells at the exposure levels observed. These results agree with some studies3,4,9,17 that demonstrated that using smartphones does not cause geno- toxicity, considering that exposed and non-exposed sides did not show statistically significant differences. However, the literature on this topic is controversial, as other studies with exfoli- ated cells5,14,18 showed a significantly higher number of micronuclei, indicating that mobile phones may cause genotoxicity in contrast with the results presented in this study. Daroit et al5. (2014) showed a slight increase in the number of micronu- cleated cells in the oral mucosa of individuals who used their phones more than 60 minutes per week over eight years. Banerjee18 et al. (2016) investigated micronuclei count in mobile phone users, comparing 150 “low-frequency users” (less than 3 h/ week using cell phones) with 150 “high-frequency users” (more than 10 h/week). Considering the “high-frequency users” group, a comparative evaluation of both sides of the buccal mucosa was performed, which showed a statistically significant higher frequency of micronuclei in exfoliated buccal cells of the exposed side than those of the contralateral side. Yadav and Sharma14 (2008) found twice as many micronuclei in mobile phone users than in non-users and reported an increased frequency of micronuclei related to the total time of exposure. However, they used orcein, a non-DNA-specific stain that may stain DNA containing micronuclei and other artifacts not associated with genomic instability, which could imply false-positive micronuclei count. The present study used hematoxylin and eosin (HE), an acid-basic stain that pro- duces a contrast between the cytoplasm and the nucleus and may mark MN fre- quency considerably19. The samples are first exposed to hematoxylin, a basic dye that binds to any substance containing acid groups, such as the phosphate groups in DNA structure, and to nuclear proteins with a negative charge. Then, samples can be exposed to eosin, a weak acid colorant that stains basic structures. The basophil structures such as nuclei are stained in blue with hematoxylin, while eosin stains acidophil structures such as collagen fibers in pink. Some complications may occur during colorant precipitation, which could facilitate a false-positive result. However, 7 Menezes et al. Braz J Oral Sci. 2022;21:e226427 if there is sufficient precaution during slide preparation and staining, this is a reliable method for MN detection15. The MN assay is often used in the oral mucosa due to rapid renewal, and the collec- tion of oral cells involves minimal invasion and high representation of the epithelial tissue4,12. The genetic analysis with exfoliated epithelial cells of the oral mucosa pro- vides several advantages because it is the primary target of exposure and the mini- mally invasive technique allows monitoring populations exposed to genotoxic agents and the association of lifestyles with the epithelial damages detected12. Accordingly, the micronucleus assay with exfoliated cells was chosen because it is well estab- lished as a reliable assessment test. There were no statistically significant differences between the brands compared. Each cell phone model has its specific absorption rate (SAR), which is the amount of energy absorbed per unit mass of tissue during a given time interval, determined by the ICNIRP3 (2009). The acceptable rate used in Brazil is two watts per kilogram of body weight5. The SAR for each device used in the study was lower than the recom- mendations of the responsible institution. Other features are directly linked to the increase in MN count. As increasing age (> 40 years) and cigarette consumption (> 40/day) exert a highly significant influence on micronucleus frequency15,20, the participants of the present study were carefully selected to exclude biases. Reducing the age difference of patients was attempted, establishing an age range of 20 to 30 years, and smoking patients were excluded. Regarding the count of the number of cells, it must be scored in order to obtain statistically results needs to be addressed. Tolbert et al.21 (1992), recommended scoring at least 1000 cells per plate, which represents a great method for determin- ing the frequency of all the various types of cells. Most recent studies, have scored between 1000 and 3000 cells, which are in accordance to the methodology adopted in our study4,9,16,19,22. A few studies3,4,17 have analyzed the potential correlation of micronuclei frequency with demographic data (sex, age, and place of birth), social origin, and environmental factors (occupation, duration and recent work changes, proximity of homes to heli- pads or airports, alcohol and tobacco consumption, diet, vitamin supplementation, family history of cancer, chronic medication, and risk factors). However, none showed statistically significant results, which agrees with most studies using the MN test for the oral mucosa. Several investigations involving the use of mobile phones are limited due to the chal- lenge to establish a control group4 because the vast majority of the population uses mobile phones, making it nearly impossible to find a sufficient number of individuals who do not use cell phones regularly. Due to this difficulty, the present study used the side of the face that was not preferred when answering calls as the control group. The side and the duration of mobile phone use are subject to errors associated with self-reporting methods because underestimations and overestimations are com- mon. Although bias is a tangible obstacle to epidemiological research, self-report- ing is often the only alternative available to evaluate certain variables. Considering the increase in the number of mobile phone users and the dilemma regarding their 8 Menezes et al. Braz J Oral Sci. 2022;21:e226427 biological consequences, the present study is important and further research is still required to better elucidate such effects. In conclusion, this study suggests that the mobile phone brands investigated do not have genotoxic potential when comparing MN frequency between the exposed buc- cal mucosa side and the non-exposed side. Data availability Datasets related to this article will be available upon request to the corresponding author. Conflict of Interests None. Author Contribution Contributor 1 Liciane Contributor 2 Itana Contributor 3 Marcos Contributor 4 Andrea Contributor 5 Silvia Contributor 6 Wilton Conception of the work √ √ √ √ Design of the work √ √ √ √ Data acquisition √ √ √ √ Data analysis √ √ √ √ √ √ Data Interpretation √ √ √ √ √ √ Manuscript preparation/ Work Draft √ √ √ √ √ √ Manuscript review/ Work Review √ √ √ √ √ √ Final approval of the version to be published √ √ √ √ √ √ References 1. Smith-Roe SL, Wyde ME, Stout MD, Winters JW, Hobbs CA, Shepard KG, et al. Evaluation of the genotoxicity of cell phone radiofrequency radiation in male and female rats and mice following subchronic exposure. Environ Mol Mutagen. 2020;61(2):276-90. doi: 10.1002/em.22343. 2. Revanth MP, Aparna S, Madankumar PD. Effects of mobile phone radiation on buccal mucosal cells: a systematic review. Electromagn Biol Med. 2020;39(4):273-81. doi: 10.1080/15368378.2020.1793168. 3. International Commission on Non-Ionizing Radiation Protection (ICNIRP). 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